WO2006080582A1

WO2006080582A1 - Agent for control of function of antigen-presenting cell

Info

Publication number: WO2006080582A1
Application number: PCT/JP2006/301933
Authority: WO
Inventors: Toshio Hirano; Hidemitsu Kitamura
Original assignee: Riken
Priority date: 2005-01-31
Filing date: 2006-01-31
Publication date: 2006-08-03
Also published as: EP1854483A4; JP2006206538A; US20090054316A1; EP1854483A1

Abstract

Disclosed is an agent for controlling the function of an antigen-presenting cell. An agent for controlling the function of an antigen-presenting cell comprising, as an active ingredient, a substance capable of controlling the concentration of zinc ions in a cell, specifically a zinc ion, a zinc-ion chelator, a substance capable of controlling the expression and/or function of a zinc ion-requiring protein or a substance capable of controlling the expression and/or function of a zinc ion transporter.

Description

Description '-Function control agent of antigen-presenting cell

Technical field

The present invention relates to a drug capable of controlling the function of antigen-presenting cells. More specifically, the present invention relates to a drug capable of controlling an immune response reaction through activation of antigen-presenting cells.

Background art

One system that maintains human health is the immune system. Control of the immune response is also extremely important from the viewpoint of vaccine development for the purpose of infection prevention, treatment of autoimmune diseases, overcoming cancer, etc., and suppression of rejection during organ transplantation. Until now, various immunostimulators such as BCG and other bacterial components have been used to control this immune response, and immunosuppressants and synthetic steroids derived from bacterial components have been administered to the human body. It was. However, these bacterial cell components and synthetic steroids have various side effects and potentially have a harmful aspect to the human body. In addition, some problems have been pointed out so far when it is used as a medicine, such as its effectiveness diminishing with long-term use.

Antigen-presenting cells such as dendritic cells are central to the control of the strength and quality of the immune response, and are being used for autoimmune diseases, allergic diseases, cancer immunotherapy, and the like. In addition, research on culture methods that control the growth and activation of antigen-presenting cells is underway. Currently, there is still a need for a method that efficiently and specifically controls the function of antigen-presenting cells. is there.

Prior art I Gutmonprez P, Val ladeau J, Zitvogel L, Thery C, Amigorena S. Antigen presentation and T cel l stimulation by denaritic cel ls. Annu Rev. Immunol. 20, 621-627 (2002), Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Palucka K. Immunobiology of dendritic cel ls.Annu Rev. Immunol. 18, 761-781 (2000), Steinman RM, ellman I.

Immunotherapy: bewitched, bothered, and bewildered no more. Science 305, 197-200 (2004). Disclosure of the invention

An object of the present invention is to provide a novel function-controlling agent for antigen-presenting cells. Specifically, the present invention controls immune responses via activation of antigen-presenting cells. The purpose is to provide a drug capable of Furthermore, the present invention applies the drug to various diseases such as treatment of autoimmune diseases, treatment of allergic diseases, suppression of rejection during organ transplantation, and further development of a method for enhancing immune response against cancer, The purpose is to develop an efficient vaccine.

As a result of intensive studies in view of the above problems, the present inventors have found that the function of antigen-presenting cells such as dendritic cells can be controlled by controlling the zinc ion level. Furthermore, it was confirmed that the function control of the antigen-presenting cell by controlling the zinc ion concentration was reversible, and the present invention was completed. That is, the present invention is as follows. [1] A function-controlling agent for antigen-presenting cells, containing as an active ingredient a substance capable of controlling intracellular zinc ion concentration.

[2] The function control agent according to [1] above, wherein the substance capable of controlling the zinc ion concentration is zinc ion.

[3] The function control agent according to the above [2], wherein the zinc ion is introduced into the cell by a zinc ionophore.

[4] The function control agent according to the above [1], wherein the substance capable of controlling the zinc ion concentration is a zinc ion killer.

[5] Zinc ion chelator is 2, 3-dimercapto-1 monopropanesulfonic acid (DMP S), N, N, Ν ', Ν, tetrakis (2-pyridylmethyl) ethylene diamine (ΤΡΕΝ), ethylene diamine (EDTA) ) And Ν- (6-Methoxy-1-8-quinolyl) -ρ-toluenesulfonamide (TSQ). The function control agent according to [4] above, which is at least one selected from the group consisting of

[6] The function control agent according to [1] above, wherein the substance capable of controlling the ffi lead ion concentration is a substance that regulates the expression and / or function of a zinc ion-requiring protein.

[7] Zinc ion-requiring protein is R af -1, PKC a, GE F— H l, H DAC, SQSTM1, ubiquitin monoprotein ligase E3, ubiquitin-coupled protein E2, metamouth thionein, phosphatase, Snai1, TRAF6, Paxi 1 1 in, Z yxin and J ade-1 The function control agent according to [6] above, which is at least one kind.

[8] The function control agent according to [1] above, wherein the substance capable of controlling the zinc ion concentration is a substance that regulates expression and Z or function of a zinc ion transporter.

[9] The function control agent according to [8] above, wherein the zinc ion transporter is at least one selected from the group consisting of human Z IPs and human CDFs including the L I V family.

[10] The antigen-presenting cell is at least one selected from the group consisting of dendritic cells, macrophages, Langerhans cells, B cells, thymic epithelial cells, synovial cells, vascular endothelial cells, and keratinocytes, [1] To [9] The function control agent according to any one of [9].

[11] The function control agent according to [10] above, wherein the antigen-presenting cell is a dendritic cell.

[1 2] A prophylactic / therapeutic agent for a disease involving an immune system, comprising the function control agent according to any one of [1] to (: 1 1) above.

[1 3] Diseases that involve the immune system consist of autoimmune diseases, allergic diseases, rejection during organ transplantation, decreased immune response due to infection and tumor formation, and abnormal immune response when using vaccines The prophylactic / therapeutic agent according to [12] above, which is at least one selected from the group.

[14] A prophylactic / therapeutic agent for diseases involving the immune system, containing as an active ingredient a substance that can control intracellular zinc ion concentration.

[15] The prophylactic / therapeutic agent according to [14] above, wherein the substance capable of controlling the zinc ion concentration is zinc ion.

[16] The prophylactic / therapeutic agent according to [15] above, wherein the zinc ion is introduced into the cell by a zinc ionophore.

[17] The substance capable of controlling the zinc ion concentration is a zinc ion chelator, [14] The preventive / therapeutic agent as described.

[1 8] The zinc ion chelator is 2,3-dimercapto-1- 1-propanesulfonic acid (DMP S), N, N, N, N, monotetrakis (2-pyridylmethyl) ethylenediamine (TPEN), ethylenediamine (EDTA) ) And N- (6-Methoxy-8-quinolyl) 1-p-toluenesulfonamide (T SQ), which is at least one selected from the group described in [17] above. medicine.

[19] The prophylactic / therapeutic agent according to [14] above, wherein the substance capable of controlling the zinc 'ion concentration is a substance that regulates the expression and / or function of a zinc ion-requiring protein.

[20] Zinc ion-requiring protein is Raf 1-1, PKCct, GEF— Η1, 'HDAC, SQSTM1, ubiquitin monoprotein ligase E3, ubiquitin-synthesizing protein E2, metamouthoneone, phosphatase, Snai1, The prophylactic / therapeutic agent according to [19] above, which is at least one selected from the group consisting of TRA F 6, Paxi 1 1 in, Z yxin and Jade-1.

[2 1] The prophylactic / therapeutic agent according to [14] above, wherein the substance capable of controlling the dumbbell ion concentration is a substance that regulates the expression and / or function of a zinc ion transporter.

[22] The prophylactic / therapeutic agent according to the above [2 1], wherein the zinc ion transporter is at least one selected from the group consisting of human Z IPs and human CD Fs containing LV family.

[23] The disease involving the immune system consists of autoimmune diseases, allergic diseases, rejection at the time of organ transplantation, decreased immune response due to infection and tumor formation, and abnormal immune response when using vaccines The prophylactic / therapeutic agent according to any one of [14] to [22], which is at least one selected.

[24] The antigen-presenting cell is at least one selected from the group consisting of dendritic cells, macrophages, Langerhans cells, B cells, thymic epithelial cells, synovial cells, vascular endothelial cells, and keratinocytes. ] The prophylactic / therapeutic agent according to any of [23].

[25] The prophylactic / therapeutic agent according to [24] above, wherein the antigen-presenting cell is a dendritic cell. [26] A method for controlling the function of an antigen-presenting cell, comprising controlling the intracellular zinc ion concentration in vitro.

[27] The method according to [26] above, wherein the zinc ion concentration is controlled by introducing zinc ions into cells.

[28] The method according to [27] above, wherein the subacute ion is introduced into the cell by a zinc ionophore.

[29] The method described in [26] above, wherein the zinc 'ion concentration is controlled by a zinc ion chelator.

[30] The zinc ion chelator is 2,3-dimercapto-1- 1-propanesulfonic acid (DMP S), N, N, Ν ', N'-tetrakis (2-pyridylmethyl) ethylenediamine (ΤΡΕΝ), ethylenediamine (EDTA) and The method according to [29] above, which is at least one selected from the group consisting of Ν— (6-methoxy-8-quinolyl) 1 ρ-toluenesulfonamide (TSQ).

[31] The method according to [26] above, wherein the zinc ion concentration is controlled by regulating the expression and function of the zinc ion-requiring protein.

[32] Zinc ion-requiring protein is Raf-1, PKCa, GEF-Hl, HDAC, SQSTM1, ubiquitin monoprotein ligase E3, ubiquitin synergistic protein E2, metamouth thionein, phosphatase, Snai1, The method according to [3 1] above, which is at least one selected from the group consisting of TRA F 6, P axil 1 in, Z yxin and J ade-1.

[33] The method according to [26] above, wherein the zinc ion concentration is controlled by adjusting the expression and / or function of a zinc ion transporter.

[34] The method according to [33] above, wherein the zinc ion transporter is at least one selected from the group consisting of human Z IPs and human CD Fs containing L IV family.

[35] From the group consisting of dendritic cells, macrophages, Langerhans cells, B cells, thymic epithelial cells, synovial cells, vascular endothelial cells and keratinocytes. The method according to any one of [2 6] to [3 4] above, which is at least one selected.

[36] A method for screening a compound capable of controlling the function of antigen-presenting cells, comprising measuring intracellular zinc ion concentration.

[37] The antigen presenting cell is at least one selected from the group consisting of dendritic cells, macrophages, Langerhans cells, B cells, thymic epithelial cells, synovial cells, vascular endothelial cells, and keratinocytes, [3 6] The method described.

[3 8] The method described in [3 7] above, wherein the antigen-presenting cell is a dendritic cell.

[39] Use of a substance capable of controlling the intracellular zinc ion concentration for producing a function control agent for antigen-presenting cells.

[40] Use of the function-controlling agent according to any one of [1] to [11] above, for producing a prophylactic / therapeutic agent for a disease involving the immune system.

[4 1] Use of a substance capable of controlling the intracellular zinc ion concentration for the manufacture of a drug for the prevention and treatment of diseases involving the immune system.

Brief Description of Drawings

FIG. 1 shows that when dendritic cells are treated with a zinc ion chelator, the expression of MH C class I, class I I, and CD 86 molecules on the cell surface is enhanced. Fig. 2 shows that when dendritic cells are treated with zinc ions and zinc ionophores, LPS-stimulated dendritic cells enhance the expression of MH CII and CD86 molecules and suppress IL-12 production. FIG.

FIG. 3 is a graph showing that dendritic cells in which m L i v 1 is expressed using a retroviral vector suppress the enhancement of MHC class I I molecule expression by L PS stimulation.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, “control of zinc ion concentration” means in addition to the literal meaning of increasing or decreasing the concentration (amount) of zinc ion in the cell, and finally the zinc ion concentration increases or decreases in the cell. Induces a phenomenon similar to that caused by In such a case, it is not restricted by the concentration of zinc ions in the cell. In the present invention, the cells to be controlled for zinc ion concentration include dendritic cells, macrophages, Lange / rehans cells, B cells, thymic epithelial cells and other antigen-presenting cells, as well as synovial cells, vascular endothelial cells, These cells are expected to acquire the ability to present antigens such as keratinocytes. Dendritic cells are preferred. In the present specification, the cells to be controlled are sometimes referred to as “antigen-presenting cells” for convenience. '

“Substances that can control intracellular zinc ion concentration” include “zinc ions”, “zinc ion chelators”, “zinc ionophores”, “substances that regulate the expression and Z or function of zinc ion-requiring proteins”, “ Examples include substances that regulate the expression and / or function of zinc ion transporters, etc.``Zinc ion J or zinc ionophore '' can directly increase the concentration of zinc ions in antigen-presenting cells by adding them. On the other hand, the “zinc ion chelator” can directly reduce the zinc ion concentration in the antigen-presenting cell by adding it, thereby controlling the function of the antigen-presenting cell. “Substances that regulate the expression and κ or function of zinc ion-requiring protein” and “Substances that regulate the expression and / or function of zinc ion transporter” regulate the expression and ζ or function of zinc ion-requiring protein. Or by regulating the expression and ζ or function of the zinc ion transporter, it is possible to control the function of antigen-presenting cells by changing the effect of zinc ions on antigen-presenting cells. . Substance that regulates the expression or function in a direction that promotes the expression or function of a substance that regulates the expression and function of a zinc ion-requiring protein or a substance that regulates the expression and / or function of a zinc ion transporter Then, the responsiveness to the zinc ion of the cell can be further enhanced, and the responsiveness to the zinc ion of the cell can be further suppressed as long as it is a substance that regulates the expression or function. In the present invention, “control” of the zinc ion concentration means both positive and negative regulation.

Antigen-presenting cells consist of 1) foreign substances (foreign substances) derived from infection, etc., and 2) self Alternatively, endogenous proteins resulting from cell tumorigenesis, virus infection, etc. should be presented to cells with TCR (eg CD4 + T cells, CD8 + T cells, DNT cells, NKT cells, etc.). Therefore, we will protect against infection and remove cells that are undesirable for ourselves by distinguishing between self and non-self. In the present invention, by controlling the function of these antigen-presenting cells, an immune response that is more favorable for the maintenance of life is elicited. “Controlling” the function of antigen-presenting cells means more specifically defining the quality and quantity of immune responses after antigen presentation. “Quality” here means response control and immune tolerance of T h 1 and T h 2 cells in CD 4+ T cells, and response of CTL with antigen-specific cytotoxic activity in CD 8 + T cells. It means control and immune tolerance, etc. “Amount J literally means strength of immune response. Also,“ controlling ”the function of an antigen-presenting cell means activation of the antigen-presenting cell itself or It means inactivation. Specifically, 1) expression control of MHC class I and II molecules, 2) expression control of costimulatory molecules such as CD 80, CD86, and CD 40, 3) IL-1, IL-6, IL-10, IL-1 2, IFN-a / j3, TNF_a, TGF—, etc., control of the expression of site force-in, 4) control of uptake of foreign or endogenous antigen, 5) control of migration ability of antigen-presenting cells, etc. Means to control the function of one or more items.

“Zinc ions” are introduced into antigen-presenting cells by zinc ionophores. In other words, zinc ions are introduced into antigen-presenting cells in the form of a complex with a zinc ionophore. Zinc ionophores include various compounds that are commonly used in the art, and preferably are commercially available. Examples include zinc pyrithione, heterocyclic amine, dithiol rubamate, and vitamins.

A “zinc ion chelator” is a substance that can remove zinc ions from cells by forming a complex with zinc ions in antigen-presenting cells. For example, 2, 3-dimercapto-1 Propanesulfonic acid (DMP S), N, N, Ν ', N' — Tetrakis (2-pyridylmethyl) Ethylenediamine (ΤΡΕΝ), Ethylenediamine (EDTA) and Ν— (6-Methoxy 8-quinolyl) Ρ-Toluenesulfonamide (T SQ) and the like. Both are commercially available. In addition, it is included as an active ingredient in the function-controlling agent of the antigen-presenting cell of the present invention, and a prophylactic / therapeutic drug for a disease involving the immune system of the present invention (hereinafter sometimes simply referred to as a prophylactic / therapeutic drug). The amount of zinc ion chelator as a substance capable of controlling the zinc ion concentration is appropriately determined within a range in which the intracellular zinc ion concentration can be controlled. One zinc ion chelator may be included, or two or more zinc ion chelators may be included. When two or more types are included, the blending amount is appropriately set according to the type.

“Substances that regulate the expression and Z or function of zinc ion-requiring protein” are not particularly limited as long as the expression and / or function of zinc ion-requiring protein can be regulated as a result. It may be regulation at the gene level or regulation at the protein level. Examples of regulation at the gene level include transcription regulation and gene expression regulation. Examples of regulation at the protein level include metabolic regulation, phosphorylation, dephosphorylation, sugar addition, lipid addition, coordinate bond with zinc, degradation, ubiquitination, acetylation, and the like. “Zinc ion-requiring protein” is a protein that requires zinc for the functional expression of antigen-presenting cells. For example, a protein having a zinc finger, a protein having a RING finger, a protein having a LIM domain, or PHD Examples include proteins having fingers. A zinc finger is a unique nucleic acid-binding motif that can have a higher-order structure only when an amino acid such as cystine-histidine is coordinated with zinc. Examples of proteins having a zinc finger include epithelium. A phenomenon called mesenchymal transition (such as body shaping in the early development of humans and other organisms, wound healing, cancer metastasis, etc.) Examples include transcription factors Snai1, PKCa, GEF—Hl, HDAC, SQSTM1, etc., which are master regulators of the phenomenon of releasing adhesion, acquiring mobility and moving to another). The RI NG finger is a variant of the zinc finger, and is suggested to be involved in protein-protein interactions, and ubiquitin monoprotein ligase E 3 (ubiquitin np roteinligase) is a protein with a RI NG finger. E 3) Active or ubiquitous It has been reported that it binds to conjugated nucleoprotein E 2 (ubiquitin—conjugating enzymes E 2) and TRAF 6 (TNF α signaling molecule). The L IM domain consists of 60 amino acids in which the positions of 6 cysteines and 1 histidine are conserved. Although it has a structure similar to that of zinc fingers, its DNA-binding ability is unknown. It also functions as a domain that mediates binding to PKC. Examples of proteins having a LIM domain include Pa'x i 11 in, Z yxin, etc. involved in cytoskeletal regulation. The PHD zinc finger is a zinc finger uniform domain, a nuclear protein that is implicated in chromatin-mediated transcriptional regulation, and is predicted to have DNA-binding ability. Examples of the protein having a PHD zinc finger include Jade-1 involved in histone acetylation.

The “substance that regulates the expression and function of zinc ion-requiring protein” may be a known compound or a new compound that will be developed in the future. Further, it may be a low molecular compound or a high molecular compound. Here, the low molecular weight compound is a compound having a molecular weight of less than about 3000, and includes, for example, organic compounds that can be normally used as pharmaceuticals and their derivatives and inorganic compounds. Derivatives, naturally-occurring compounds and derivatives thereof, small nucleic acid molecules such as promoters, various metals, and the like, desirably organic compounds that can be used as pharmaceuticals, derivatives thereof, and nucleic acid molecules. The polymer compound is a compound having a molecular weight of about 3000 or more, and includes proteins, polynucleic acids, polysaccharides, and combinations thereof, and is preferably a protein. These low-molecular compounds or high-molecular compounds are commercially available if they are known, or can be obtained through steps such as collection, production, and purification according to each report literature. These may be of natural origin, prepared by genetic engineering, or obtained by semisynthesis. Specifically, the expression of zinc auxotrophic protein S nai 1 is regulated by MAPK through TGF-] 3 or FGF (Peinado, H., Quintanilla,. & Cano, A. Transforming growth factor beta-1 induces snail transcription factor in epithelial cell lines: mechanisms for epithelial mesenchymal transitions. J. Biol. Chem. 278, 21113-23 (2003)). Snai 1 is activated by LIV 1 (described later), which is a zinc ion transporter (Yamashita, S., Miyagi, C., Fukada, Τ ·, Kagara, Ν., Che, Υ, -S. & Hirano, Τ. Zinc transporter LIVI controls

epithelial-mesenchymal transition in zebraf ish gastru 丄 a organizer. Nature 429, 298-302 (2004)). For other zinc ion-requiring proteins, Raf 1, GEF— Η ί, PKC, phosphatase, and meta-mouthonein are also available. Jirakulaporn T, Muslin AJ. Cation diffusion racilitator proteirrs modulate Raf-1 activity. J Biol. Chem. 279, 27807-15 (2004) _N Krendel M, Zenke FT, Bokoch GM.Nucleotide exchange factor GEF-HI mediates cross-talk between microtubules and the actin cytoskeleton.Nature Cell Biology 4, 294-301 (2002) ), Korichneva I, Hoyos B, Shihua R, Levi E, Hammer ling U. Zinc release from protein kinase C as the common event during activation by lipid second messenger or reactive oxygen. J. Biol. Chem. 277, 44327-31 (2002), Haase H, Maret W. Intracellular zinc fluctuations modulate protein tyrosine phosphatase activity in insulin / insulin-like growth factor-1 signaling. Exp. Cell Research 291, 289-298 (2003), Biochemical Dictionary (3rd edition) ), Published by Tokyo Chemical Doujin, page 1 393 (1 998) for details. For TRAF 6, P axillin, Z yxin, J ade— 1 Kobayashi, Kadono Y, Naito A, Matsumoto K, Yamamoto T, Tanaka S, Inoue J. Segregation of TRAF6-mediated signaling pathways clarities its role in osteoclastogenesis. EMB0 J. 20, 1271-1280 (2001), Brown MC, Perrotta JA, Turner CE.

Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding.J. Cell Biol. 135, 1109-1123 (1996), Sadler I, Crawford AW, Michelsen JW , Beckerle MC. Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton.J. Cell Biol.119, 1573-1587 (1992), Panchenko MV, Zhou MI, Cohen HT.von Hippel-Lindau partner Jade-1 is a transcriptional co-activator details are described in associated with histone acetyl transferase activity. J. Biol. Chem. 279, 56032-56041 (2004). In addition, SQS TM 1 and HD AC can be used as an example Joung I, Strominger JL, Shin J. Molecular cloning of a phosphotyrosine-independent ligand of the p561ck SH2 domain. Proc Natl Acad Sci USA, 93, 5991-5995 ( 1996) and Kawaguchi Y, Kovacs JJ, McLaurin A, Vance JM, Ito A, Yao TP.The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress.Cell 115, 727-738 (2003). Has been.

“A substance that regulates the expression and / or function of zinc ion transporter” means that the mechanism of action is not particularly limited as long as the expression and Z or function of zinc ion transporter can be regulated as a result. It may be at the level or at the protein level. Examples of regulation at the gene level include transcription regulation and gene expression regulation. Examples of regulation at the protein level include metabolic regulation, phosphorylation, dephosphorylation, sugar addition, lipid addition, coordinate bond with zinc, degradation, ubiquitination, acetylation, and the like. “Zinc ion transporters” include, for example, human ZIPs containing LIV family (BAB 70848, h Z ip 4, BI GM1 03, KI AA006 2, KI AA1 265, h L iv—1, A AH 088 53, hKE 4, XP— 20864 9, h ZIP l, h ZIP 2, h ZIP 3, BAA 9 2 100, BAC04504, etc., human CDFs (h Z nt _ 5, h Z nt— 7 HZnt-1, 1, hZnt-6, hZnt-3, hZnt-8, hZnt-4, hZnt-9, etc.). LIV 1 was originally identified as an estrogen-controlled breast cancer protein, and recently the Zinc ion transporter subfamily (Z rt) called LZT (LIV 1 subfamily of ZIP zinc ion transporters). ― I rt (Taylor, KM & Nicholson, RI The LZT proteins; the LIV-1 subfamily of zinc transporters. Biochim. Biophys. Acta 1611, 16-30 (2003)) and function as a sub-ion ion transporter (Taylor, K.., Morgan, HE, Johnson, A., Hadley, Shi J. & Nicholson, RI

Structure-function analysis of LIV-1, tne breast cancer-associated protein that belongs to a new subfamily of zinc transporters. Biochem. J. 375, 51-9 (2003)). Other examples include C DF (c a t i o n d i f f u s i o nf a c i 1 i tat o r) and the like. For example, the zinc ion transporter LIV 1 is regulated by STAT 3 (Yamashita, S., iyagi, C., Fukada, Τ. ', Kagara, N., Che, Y.-S. & Hirano , T. Zinc transporter LIVI controls epithelial-mesenchymal transition in zebraf ish gastrula organizer. Nature 429, 298-302 (2004)).

The “substance that regulates the expression and function of zinc ion transporter” may be a known compound or a new compound to be developed in the future. Further, it may be a low molecular compound or a high molecular compound. Here, the low molecular weight compound is a compound having a molecular weight of less than about 300, for example, an organic compound that can be usually used as a pharmaceutical, a derivative thereof, and an inorganic compound. Compounds, derivatives thereof, naturally derived compounds, derivatives thereof, small nucleic acid molecules such as promoters and various metals, and preferably organic compounds that can be used as pharmaceuticals, derivatives thereof, and nucleic acid molecules. The polymer compound is a compound having a molecular weight of about 300 or more, and includes proteins, polynucleic acids, polysaccharides, and combinations thereof, and is preferably a protein. These low-molecular compounds or high-molecular compounds are commercially available if they are known, or can be obtained through steps such as collection, production, and purification according to each report literature. These may be of natural origin, prepared by genetic engineering, or obtained by semisynthesis.

LIV 1, a zinc ion transporter, is a zinc ion-requiring protein W

14 Because it enhances the activity of S nai 1 (especially repressor activity) (Yama ^ hita, S., Miyagi, C., Fukada, X., Kagara, N., Che, Y.— S. & Hirano, T. Zinc transporter LIVI controls epithelial-mesenchymal transition in zebraf ish gastrula organizer.Nature 429, 298-302 (2004)), `` Substances that regulate the expression and Z or function of subthin requirement protein '' in the present invention The following are examples.

(1) DN A as described in any of a) to d) below

a) DNA encoding a protein having the amino acid sequence described in SEQ ID NO: 2, 4 or 6 (LIV 1 protein)-b) DNA comprising the sequence described in SEQ ID NO: 1, 3 or 5 (LIV 1 gene) c) DNA encoding a protein (protein similar to LIV 1) having an amino acid sequence in which one or more amino acid sequences are substituted, deleted, inserted and Z or added in the amino acid sequence described in SEQ ID NO: 2, 4 or 6

d) DNA that hybridizes with the sequence of SEQ ID NO: 1, 3 or 5 under stringent conditions

(2) A vector into which the DNA according to any one of a) to d) below is inserted

a) DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2, 4 or 6

b) DN A consisting of the sequence described in SEQ ID NO: 1, 3 or 5

c) DNA encoding a protein having an amino acid sequence in which one or more amino acid sequences are substituted, deleted, inserted and Z or added in the amino acid sequence described in SEQ ID NO: 2, 4 or 6

d) DNA that hybridizes with the sequence described in SEQ ID NO: 1, 3 or 5 under stringent conditions

(3) A protein encoded by the DNA according to any one of a) to d) below

a) encodes a protein having the amino acid sequence of SEQ ID NO: 2, 4 or 6 DNA

b) DNA consisting of the sequence described in SEQ ID NO: 1, 3 or 5

c) DNA encoding a protein having an amino acid sequence in which one or more amino acid sequences are substituted, deleted, inserted and Z or added in the amino acid sequence of SEQ ID NO: 2, 4 or 6

d) DNA 'that hybridizes with the sequence described in SEQ ID NO: 1, 3 or 5 under stringent conditions.

(4) Antisense oligonucleotides whose target sequence is DNA consisting of the sequence described in SEQ ID NO: 1, 3 or 5

(5) Double-stranded RNA having a sequence identical or similar to a part of DNA consisting of the sequence described in SEQ ID NO: 1, 3 or 5

(6) An antibody against a protein having the amino acid sequence set forth in SEQ ID NO: 2, 4 or 6

(7) Substances that associate with the protein having the amino acid sequence of SEQ ID NO: 2, 4 or 6 and regulate its function (including natural products and non-natural products)

(1) to (3) are substances that positively regulate the expression and / or function of Snai 1 which is a zinc ion-requiring protein (enhance expression and activate Z or function) (4 ) And (5) are substances that negatively regulate the expression and / or function of Snai 1 (reduce expression and / or suppress function).

The LIV 1 protein can be prepared by various methods well known to those skilled in the art. For example, it can be prepared by producing and purifying a protein in a transformant holding a vector having the DNA (LVI gene) having the nucleotide sequence set forth in SEQ ID NO: 1, 3 or 5 inserted therein. The vector to be used can be appropriately selected depending on the translation system used for protein production. LIV 1 is known to be expressed in hormonal tissues such as breast, prostate, pituitary gland, and brain (Taylor, KM & Nicholson, R. 丄. The LZT proteins; the LIV-1 subfamily of zinc transporters. Biochim. Biophys. Acta 1611, 16-30 (2003)). Around anti-LIV 1 protein antibody LIV 1 protein can be purified from a cell extract expressing LIV 1 by preparing it by an intelligent method and preparing a affinity column with the antibody.

A protein similar to LIV 1 is a protein capable of regulating the activity of a zinc ion-requiring protein (for example, S nai 1), for example, one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2, 4 or 6 DNA that hybridizes under stringent conditions with a protein consisting of an amino acid sequence with substitution, deletion, insertion, and addition or deletion, or with a DNA consisting of a base sequence according to SEQ ID NO: 1, 3 or 5 The protein encoded by can be mentioned.

For the preparation of a protein similar to LIV 1, for example, hybridization is performed using a base sequence encoding LIVI, a transformant is prepared using the obtained highly homologous DNA, A method can be used in which the transformant produces the desired protein. As an example of a method for obtaining highly homologous DNA, a part of the base sequence described in SEQ ID NO: 1, 3 or 5 is used as a probe, and stringent hybrids are obtained from cells of vertebrate animals other than human rabbits. An example is a method of hybridizing under dithering conditions.

Those skilled in the art can appropriately select the stringent hybridization conditions. For example, a hybrid solution containing 25% formamide, 50% formamide under more severe conditions, 4 XS SC, 50 mM Hepes pH 7.0, 10 X Denhardt's solution, 20 8 11 and denatured salmon sperm 0 After prehybridization at 42 ° C, add labeled probe and incubate at 42 ° C for high prehybridization. In the subsequent cleaning, the cleaning solution and temperature conditions are about `` 1 XSSC, 0.1% SDS, 37 ° C '', and more severe conditions are `` 0.5 XSSC, 0.1% SDS, 42 ° CJ, More severe conditions can be implemented at “0.2 XS SC, 0.1% SDS, 65 ° C”. Thus, isolation of DNA having high homology with the probe sequence can be expected as the conditions for washing hybridization are more severe. The combination of SSC, SD S and temperature conditions is an example, A person skilled in the art can appropriately combine the above or other factors (for example, probe concentration, probe length, hybridization reaction time, etc.) that determine the stringency of the hybridization. It is possible to achieve the same stringency as

A polypeptide encoded by DNA isolated using such a hybridization technique usually has a high homology in amino acid sequence with L I V 1. High compatibility means at least 40% or more, preferably 60% or more, more preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 97% or more. This refers to the sequence homology (for example, 98 to 99%). The identity of amino acid sequences can be determined by, for example, the algorithm B LAST (Proc. Natl. Ac ad. S ci. USA 8 7: 2264-2268, 1 990, Proc. Na tl. Ac a d. S c in USA 90: 587 3-58 77, 1 9 3 3). Based on this algorithm, a program called B LAS TX has been developed (Alt s c hul eta 1. J. Mo 1. B i o l. 2 1 5: 40 3— 4 10, 1 990). When analyzing the amino acid sequence by B LAS TX, the parameter 1 is, for example, s corre = 50 and wo rd lenght = 3. When using BLAST and Gappe ED BLAST programs, use the default parameters of each program. Specific methods of these analysis methods are publicly known, http: ZZwww.ncbi.n1m.nih.gov.).

Preparation of a protein similar to L I V 1 can also be performed by other known means. For example, it is possible to artificially modify LIVI DNA by site-directed mu tagenesis such as deletion mu tant production method using exonuclease and cassette mutation method, and to prepare desired protein using the modified LIV 1 DNA. it can.

Another preferred example of a substance that can be used as a substance that regulates the expression and Z or function of a zinc ion-requiring protein is the sequence described in SEQ ID NO: 1, 3, or 5. DNA. The above DNA encodes the protein LIV 1, and if introduced into a cell, it is a substance that can indirectly adjust the activity of a zinc-requiring protein, such as Snai 1, through LIV 1 protein expression. .

The above DNA should be prepared from cDNA of cells expressing LIV 1 by a hybridization technique well known to those skilled in the art using a part of the sequence shown in SEQ ID NO: 1, 3 or 5 as a probe. Can do. It can also be obtained by performing RT-PCR from mRNA using a part of the sequence shown in SEQ ID NO: 1, 3 or 5 as a primer. Alternatively, it may be artificially synthesized using a commercially available DNA synthesizer.

Furthermore, DNA similar to the above DNA is also an example of “a substance that regulates the expression and function of a zinc ion-requiring protein”. The similar DNA is a DNA that hybridizes under stringent conditions with the sequence shown in SEQ ID NO: 1, 3, or 5, and is a substance that regulates the expression and / or function of a zinc ion-requiring protein, for example, A DNA encoding a protein capable of regulating the activity of Snai 1 can be mentioned. The method for preparing this DNA is as described above.

When using each DNA described above, it can be used by inserting it into an appropriate vector. A product inserted into a vector is also one embodiment of the present invention. The vector to be used can select an appropriate vector according to the purpose. Specifically, vectors derived from mammals (for example, pc DNA 3 (manufactured by Invitrogene) and pEG F- BOS (Nucleic Acids. Res., 1 8 (17), p. 5 32 2, 1 990), p EF, p CDM8, p CXN), insect cell-derived vectors (eg “B ac-to- BAC baculovirus expression system” (Invitrogen), p B ac PAK8), plant-derived expression vectors 1 (for example, ρΜΗ1, pMH2), a vector derived from an animal virus (for example, p HSV, pMV, pAd ex L cw), a vector derived from retrowinoles (for example, p ZIP neo), a vector derived from yeast (for example, “P ichia E xpression Kit J (manufactured by Invitrogen), pNV ll, SP—Q0 1), vectors derived from Bacillus subtilis (eg, p PL 608, p KTH 50), E. coli vector (M 1 3 Vector vectors, pUC vectors, pBR322, pBluescript ^ pCRScript) and the like. In the present invention, it is preferable to use one vector that can be expressed in mammalian cells, and it is also preferable to use an expression vector. In order to introduce a vector into cells, for example, calcium phosphate method (Virology, Vol. 52, p. 456, 1 973), DEAE dextran method, Cationic ribosome DOTAP (Roche. Diagnostics) , Electo mouth position method (Nuc 1 eic Acids Res., Vo l. 1 5, p. 1 3 1 1, 1 98 7), lipofection method (J. C 1 B ioch em. Nutr., Vo l. 7, p. 1 75, 1 98 9), virus introduction method (pMX, pMSCV, etc .; S ci. Am., p. 34, 1 994) ), By selecting from a particle gun or the like.

“Substances that regulate the expression and Z or function of a zinc ion-requiring protein”, for example, an antisense oligonucleotide that targets DNA or mRNA encoding LIV 1 as an example of an agent that suppresses the activity of Snai 1 be able to. Antisense oligonucleotides to DNA encoding L I V 1 are thought to prevent expression of the endogenous L I V 1 gene and negatively regulate Snai 1 activity. Examples of such antisense oligonucleotides include antisense oligonucleotides having a DNA sequence consisting of the sequence set forth in SEQ ID NO: 1, 3 or 5 or an mRNA sequence generated from the DNA sequence as a target sequence. As an example, the oligonucleotide described in SEQ ID NO: 7 or 8 can be mentioned. In addition to this, it is included in the antisense oligonucleotide of the present invention as long as it hybridizes to any location such as DNA consisting of the sequence set forth in SEQ ID NO: 1, 3 or 5 and can effectively inhibit LIV 1 expression. It does not have to be completely complementary to DNA consisting of the sequence set forth in SEQ ID NO: 1, 3 or 5 or the corresponding mRNA.

The antisense oligonucleotide can be used by inserting it into an appropriate vector depending on the purpose. For example, for the purpose of gene therapy, retrovirus vector, adenovirus vector, vaccinia virus vector, etc. It can be appropriately selected from viral vectors, non-viral vectors such as cationic ribosomes and ligand DNA complexes. It is also conceivable to administer a large volume of aqueous solution as naked plasmid DNA without using a carrier.

Another “substance that regulates the expression and function of zinc ion-requiring protein”, particularly as an example of an agent that suppresses the activity of Snai 1, a sequence identical or similar to a portion of DNA encoding LIV 1 And a double-stranded RNA having Double-stranded RNA having a sequence that is the same as or similar to the target gene sequence can cause RNA interference (RNAi) that prevents the expression of the target gene. RNA i refers to a phenomenon in which when a double-stranded RNA (dsRNA) is introduced into a cell, the mRNA in the cell corresponding to the RNA sequence is specifically degraded and is not expressed as a protein. The region that forms a double strand may form a double strand in all regions, or a part of the region (for example, both ends or one end) may be a double strand or the like. Good. Therefore, the double-stranded RNA of the present invention may contain a region that is not double-stranded. Oligo RNA used for RNA i is often 10 to: L 00 bp RNA, and usually 19 to 23 bp RNA. RNAi method is Nature, Vo l. 39 1, p. 806, 1 9 98, Proc. N atl. Ac ad. S ci. USA Vo l. 95, p. 1 5502, 1 998, N ature, Vol. 395, p. 854, 1 998, Proc. Natl. A cad. S ci. USA Vol. 96, p. 5049, 1 99 9, Cell, Vol. , p. 1 0 1 7, 1 998, Proc. N at 1. Ac a d. S ci. USA Vo l. 96, p. 1 45 1, 1 9 99, Proc. N atl. Ac a d. Sci. U SA Vol. 95, p. 1 3 9 59, 1 998, Nature Cell Biol., Vol. 2, p. 70, 2000, etc.

As a “substance that regulates the expression and / or function of a zinc ion transporter”, for example, when the substance is LIV 1, Examples are the same as “substances that regulate expression and Z or function”.

When the “Zinc ion transporter” is other than LIV 1, for example, h Z nt— 1, h Z nt— 2, h Z nt— 5, h Z nt— 6, h Z nt— 7, h ZIP l, h ZIP 2, h ZIP 3, BAA 9 2 1 00, BAC 04504, h L iv— 1, hKE 4, KI AA 1 265, KI AA 0062, h Z ip 4 Based on the known amino acid sequence or base sequence, various “substances that regulate the expression and / or function of zinc ion transporter” or “expression and expression of zinc ion-requiring protein” as in LIV 1. A substance that modulates Z or function can be prepared. -Information on various amino acid sequences and nucleotide sequences can be obtained from various databases that can be browsed by NCB I.

The agent for controlling the function of antigen-presenting cells and the prevention of diseases involving the immune system according to the present invention includes the above-described series of active ingredients (zinc ion, zinc ionophore, zinc ion killer, expression of zinc ion-requiring protein) And substances that regulate Z or function, substances that regulate the expression and function of zinc ion transporter), and optionally include pharmaceutically acceptable excipients and additives. Pharmaceutically unacceptable excipients and additives include carriers, binders, fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc. Can be mentioned. Also, different types of “substances that control zinc ion concentration” can be used in combination as active ingredients.

Examples of pharmaceutically acceptable carriers include magnesium carbonate, magnesium stearate, tanolec, sugar, ratatoose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and low melting point. Examples include wax and cocoa butter.

Furthermore, the tablets can be made into tablets with ordinary coatings as necessary, for example, sugar-coated tablets, enteric-coated tablets, film-coated tablets, double-layer tablets, and multilayer tablets. Powders are formulated with a pharmaceutically acceptable powder base. Examples of bases include talc, lactose, and starch. Drops are aqueous or non-aqueous It can be formulated together with a base and one or more pharmaceutically acceptable diffusing agents, suspending agents, solubilizing agents and the like. Capsules can be produced by filling a compound as an active ingredient together with a pharmaceutically acceptable carrier. The compounds can be mixed with pharmaceutically acceptable excipients or filled into force capsules without excipients. A cachet agent can be produced by a similar method. When the present invention is prepared as a suppository, it is usually used together with a base such as vegetable oil (castor oil, olive oil, peanut oil, etc.), mineral oil (petrol, white petrolatum, etc.), waxes, 'partially synthesized or fully synthetic glycerin fatty acid ester. Formulated according to the technique used.

Examples of injection solutions include solutions, suspensions, and emulsions. For example, an aqueous solution, a water-propylene glycol solution, etc. are mentioned. The solution can also be produced in the form of a solution of polyethylene and / or propylene dalicol, which may contain water.

A solution suitable for oral administration can be produced by adding a compound as an active ingredient to water and adding a colorant, a fragrance, a stabilizer, a sweetener, a solubilizer, a thickener and the like as necessary. A solution suitable for oral administration can also be produced by adding the compound together with a dispersant to water to make it viscous. Examples of the thickener include pharmaceutically acceptable natural or synthetic gum, resin, methylcellulose, sodium carboxymethylcellulose, or a known suspending agent.

Examples of the topical administration agent include the above-mentioned liquids, creams, aerosols, sprays, powders, lotions, ointments and the like. The above-mentioned topical preparation can be produced by mixing a compound as an active ingredient with a pharmaceutically acceptable diluent and carrier. Ointments and creams are formulated, for example, by adding a thickener and a Z or gelling agent to an aqueous or oily base. Examples of the base include water, liquid paraffin, and vegetable oil. Examples of the thickening agent include soft paraffin, anolenomium stearate, cetostellenoreanolol, propylene glycolate, polyethylene glycol, lanolin, hydrogenated lanolin, and beeswax. For topical administration, if necessary, methyl hydroxybenzoate, propyl hydroxybenzoate, Preservatives such as black mouth cresol and benzalkonium chloride and bacterial growth inhibitors can be added. Lotions can contain one or more pharmaceutically acceptable stabilizers, suspending agents, emulsifiers, diffusing agents, thickeners, colorants, flavors, etc., in an aqueous or oily base. .

The antigen-presenting cell function-controlling agent of the present invention and the prophylactic / therapeutic agent for diseases related to the immune system are administered orally or parenterally.

When administered orally, it can be administered in a dosage form commonly used in the art. When administered parenterally, it can be administered in a dosage form such as a topical administration agent (transdermal agent, etc.), a rectal agent, an injection, or a nasal agent.

Examples of the oral preparation or rectal preparation include capsules, tablets, pills, powders, drops, cachets, suppositories, and liquids. Examples of injections include sterile solutions or suspensions. Examples of topical agents include creams, ointments, lotions, transdermal agents (ordinary patches, matrix agents) and the like.

The dose and the number of doses vary depending on the type of substance that can control the zinc ion concentration to be used, the patient's symptoms, age, weight, dosage form, etc., and can be appropriately set.

"Zinc ions", "Zinc ion chelators", "Substances that regulate the expression and / or function of zinc ion-requiring proteins" and "Substances that regulate the expression and Z or function of zinc ion transporters" are all In mammals such as humans, horses, horses, dogs, mice, rats, etc., the function of antigen-presenting cells, specifically the expression of molecules related to antigen presentation, the expression of antigen-presenting cells by LPS, etc. It is possible to control activation, site force-in production, antigen uptake, migration ability, etc., and therefore prevent diseases related to the function of antigen-presenting cells, specifically diseases related to the immune system It can be treated. Diseases that involve the immune system include autoimmune diseases, allergic diseases, cancer, rejection during organ transplantation, decreased immune response due to infection and tumor formation, and abnormal immune responses (eg, side effects) when using vaccines. Can be mentioned. For example, the present invention includes SIRS (systemic inflammatory response syndrome), systemic lupus erythematosus, mixed connective tissue disease, rheumatoid arthritis, siedalene syndrome, rheumatic fever, Good Pastier I syndrome, Graves' disease, Hashimoto's disease, Addison's disease, autoimmune hemolytic anemia, idiopathic platelet-poor purpura, myasthenia gravis, ulcerative colitis, Crohn's disease, interchangeable ophthalmitis, multiple sclerosis To treat psoriasis, anaphylactic shock, allergic rhinitis, allergic conjunctivitis, bronchial asthma, measles, atopic dermatitis, etc. It is expected that it can be used for post-administration and for controlling immune responses against cancer. Cancers include breast cancer, prostate cancer, knee cancer, stomach cancer, lung cancer, colon cancer (colon cancer, rectal cancer, anal cancer), esophageal cancer, duodenal cancer, head and neck cancer (tongue cancer, pharyngeal cancer), Brain tumor, schwannoma, non-small cell lung cancer, small cell lung cancer, liver cancer, kidney cancer, bile duct cancer, uterine cancer (uterine body cancer, cervical cancer), ovarian cancer, bladder cancer ', skin cancer, hemangioma, Malignant lymphoma, Malignant melanoma, Thyroid cancer, Bone tumor, Hemangioma, Angiofibroma, Retinal sarcoma, Penile cancer, Pediatric solid cancer, Force positive sarcoma, Capodisarcoma due to AIDS, Maxillary sinus tumor, Fibrous histiosphere Tumor, leiomyosarcoma, rhabdomyosarcoma cancer and malignant tumor such as leukemia.

In addition, by introducing zinc ions into antigen-presenting cells using a drug such as zinc ionophore, it becomes possible to suppress activation of antigen-presenting cells by LPS or the like, and to suppress site-in production. Furthermore, it is possible to control the balance of T h 1 and T h 2 and the activation of CTL and B cells. In other words, the agent for preventing and treating diseases associated with the function-controlling agent of the antigen-presenting cell and the immune system of the present invention suffers from various diseases whose therapeutic effect is recognized by the introduction of zinc ions into the antigen-presenting cell. Patients can also be the subject of administration.

Furthermore, based on the knowledge obtained by the present inventors that the function of antigen-presenting cells can be controlled by controlling the zinc ion concentration, antigen-presenting cells can be measured by measuring the intracellular zinc ion concentration. It is possible to screen for compounds that can control the functions of these. For example, the screening method is implemented by the following steps.

1) Dividing antigen-presenting cells into two groups and treating one with the test compound (the remaining untreated is the control) 2) A step of measuring the intracellular zinc-on concentration of treated cells and untreated control cells.

3) A step of selecting a compound whose zinc ion concentration is significantly different from that of the control cell and certifying it as a compound capable of controlling the function of the antigen-presenting cell.

As the antigen-presenting cells, those described above are used. Dendritic cells are preferred. Appropriately divide into two groups and treat one with the test compound. The other is not treated and used as a control cell. Furthermore, compounds known to affect the zinc ion concentration, such as zinc ion chelator TPEN, may be used as a positive control compound. The test compound may be a known compound or a new compound to be developed in the future. Further, it may be a low molecular compound or a high molecular compound. Here, the low molecular weight compound is a compound having a molecular weight of less than 300, for example, an organic compound that can be normally used as a pharmaceutical, a derivative thereof, and an inorganic compound. Compounds, derivatives thereof, naturally derived compounds, derivatives thereof, small nucleic acid molecules such as promoters and various metals, and preferably organic compounds that can be used as pharmaceuticals, derivatives thereof, and nucleic acid molecules. The high molecular compound is a compound having a molecular weight of about 300 or more, and includes proteins, polynucleic acids, polysaccharides, and combinations thereof, and is preferably a protein. These low-molecular compounds or high-molecular compounds can be obtained commercially if they are known, or can be obtained through steps such as collection, production, and purification according to each report literature. These may be of natural origin, may be prepared by genetic engineering, or may be obtained by semisynthesis or the like.

The treatment time of the cells with the test compound is appropriately set depending on the type of cells used and the test compound. In the case of using a positive control compound such as TPEN, it can be carried out with reference to the fact that fluctuations in the zincion concentration are confirmed when treated with the control compound. The concentration of the test compound for treating the cells is also set appropriately depending on the type of cells used, the type of test compound, and the treatment time. The measurement of intracellular zinc ion concentration can be carried out by using a method usually carried out in this field or according to it. For example, it can be measured directly by an atomic absorption method (frame method) or by a fluorescence spectrophotometer using a specific probe (for example, a fluorescent reagent).

Example

Hereinafter, the present invention will be described in more detail with reference to examples, but these examples do not limit the scope of the present invention in any way. All publications cited throughout this application are hereby incorporated by reference. In addition, the reagents, devices, and materials used in the present invention are commercially available unless otherwise specified.

Example 1

MHC class on the cell surface when dendritic cells, one of the antigen-presenting cells, are treated with the zinc ion chelator N, N, N, N '—tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) The expression status of I molecule, class II molecule and CD 86 molecule was examined.

Bone marrow cells were obtained from C 5 7 B LZ6 mice (obtained from 3 Claires), 10% FC S (EQU I TECH-B IOInc., # S FB—30— 1 388), granulocytes, macrophage colonies Cells were cultured in RPMI medium (SI GMA, R 87 58) with stimulating factor (GM—CSF) for 6 days at 37 ° C in a 5% CO ₂ environment, and bone marrow-derived dendritic cells (BMDC) were cultured. Differentiation was induced. To this BMDC, TPEN (SI GM A, P 44 1 3) was added to the medium at a concentration of 0 to 2 M for 6 to 12 hours. At the same time, a group with addition of lipopolysaccharide (LPS; 10 Ong / ml; SI GMA, L 26 37, E. coli O 55: B 5) was also prepared as a condition for controlling the activation of antigen-presenting cells. did. 1 MHC class I, class II and CD86 molecules that had been expressed on the cell surface after 2 hours (or 6 hours) were evaluated by FACS analysis. The results are shown in Figure 1. The numbers in Fig. 1 indicate the percentage of cells that highly express each molecule. Treatment with a zinc ion chelator enhanced the expression of MHC class II molecules on BMDC in the same manner as LPS. This enhanced expression Was observed in a dose-dependent manner (treatment time 12 hours) and treatment time-dependent (treatment concentration .5 IX M).

Example 2

BMDC prepared in the same manner as in Example 1 was added with zincthiophore pyrithione (5 / M; Mollecularprobes, ρ—24 1 93) and Zn ²⁺ (Z n S0 ₄ ) in the final concentration of 5 In addition, it was stimulated with LPS (10 OngZmL '). Six hours later, MHC class II and CD86 molecules expressed on the cell surface were evaluated by FACS analysis. The result is shown in figure 2. The numbers in Figure 2 indicate the percentage of cells that highly express each molecule. At the same time, IL-12 production of BM DC was immobilized with 4% paraformaldehyde, followed by anti-IL-12 p40 rod (BD. Bioscience P harmingen, # 5 54 4 7 9) and isotype control antibody. (BD. Bioscience P harmingen, # 5 5 3 9 2 5) was used for detection by intracellular staining. The numbers in the figure show the percentage of cells producing IL-12.

The expression of MHC I I and CD 86 molecules on the cell surface and the production of IL-12 cytokines following activation of BMDC by LPS stimulation were not observed in the presence of zinc ionophore.

In the same experiment as in Examples 1 and 2 (in Vitro system), activation or inactivation of dendritic cells was observed by treatment with 1 to 10 μΜ of zinc ions or T P EN.

Example 3

Using a retroviral vector that expresses mouse Li V 1 as a substance that controls zinc ion concentration, the effect of LPS stimulation on the enhancement of MHC class I I molecule expression was examined.

Restriction enzymes X ho I and Not from clones (mfj 046 2 3) containing the mouse Li V 1 gene (mL iv 1, gene sequence: SEQ ID NO: 5, amino acid sequence: SEQ ID NO: 6) provided by RIKEN The mL i V 1 gene was cleaved and collected by I, and the University of Tokyo Medical Research Institute Produced by Prof. Toshio Kitamura and incorporated into the provided p MX—I RE S—GF P retrovirus vector. This pMX—IRE S-GF P-mL iv 1 vector was introduced into Phoenix cells provided by Dr. Garry P. No 1 an at Stanford University, USA using lipofectamine (Invitrogen) to transfer the virus. Expressed. After 24 to 48 hours, the culture supernatant containing this virus was collected, and GM-CSF [GM-CSF producing cell line (GM-CSF / CHO) was cultured, and the culture supernatant was treated with 0.3% FCS (10%) and prot am inesu 1 fate (SI GMA; 2 μg / ml) were added to obtain a virus infection solution. C 5 7 B LZ6 mouse (S-crea) BMDC prepared from bone marrow was infected with BM DC by replacing the medium with virus infection solution in Day 2 and Day 4. The medium was replaced with virus-free medium with Day 6, and further stimulated with LPS (SI GMA; 100 ng / mL). Cells were collected 6 hours after stimulation, and the expression level of MHC class II molecules on the cell surface was examined by FACS. Here, a cell group not stimulated with LPS was treated as a control, and a GFP positive cell was treated as a cell in which mL i V 1 was forcibly expressed.

It was found that the enhanced expression of MHC class I I molecules by L PS stimulation was suppressed in cells with forced expression of m L i v 1.

(Sequence listing free text)

Sequence number 7: Antisense sequence | arrangement

Sequence number 8: Antisense sequence | arrangement

Industrial applicability

In antigen-presenting cells, molecules involved in antigen presentation (eg, MHC class I, class) by controlling the zinc ion concentration or regulating the expression and function of zinc ion-requiring proteins and zinc ion transporters II, CD 86 molecule, etc.) can be enhanced. Furthermore, introduction of zinc ions into antigen-presenting cells with a drug such as zinc ionophore can suppress activation of antigen-presenting cells and production of cyto force in by lipopolysaccharide (LPS). Follow Therefore, it is only necessary to regulate the intracellular level of zinc originally present in the body without using conventional immunostimulatory molecules or immunosuppressive agents such as bacterial components or synthetic steroids. Thus, for example, it is possible to enhance or suppress the immune system through activation of antigen-presenting cells such as dendritic cells. This application is based on a Japanese patent application, Japanese Patent Application No. 2005-0223200 (filing date: Jan. 3, 2005) ', the entire contents of which are included in this specification.

Claims

The scope of the claims , '

1. a function-controlling agent for an antigen-presenting cell, which contains, as an active ingredient, a substance capable of controlling intracellular zinc ion concentration.

2. The function control agent according to claim 1, wherein the substance capable of controlling the zinc ion concentration is zinc ion.

3. The function control agent according to claim 2, wherein the zinc ion is introduced into the cell by a zinc ionophore.

4. The function control agent according to claim 1, wherein the substance capable of controlling the zinc ion concentration is a zinc ion killer.

5. Zinc ion chelator is 2, 3-dimercapto 1-propanesulfonic acid (DMP S), N, N, Ν ', N'-tetrakis (2-pyridylmethyl) ethylene diamine (ΤΡΕΝ), ethylene diamine (EDTA) ) And Ν- (6-Methoxy 8-quinolyl) 1 ρ-Toluenesulfonamide (T SQ). 5. The function control agent according to claim 4.

6. The function control agent according to claim 1, wherein the substance capable of controlling the zinc ion concentration is a substance that regulates the expression and / or function of a zinc ion-requiring protein.

7. Zinc ion-requiring proteins are Raf-1, PKCa, GEF—H1, HD AC, SQSTM1, ubiquitin monoprotein ligase E3, ubiquitin-conjugated protein E2, metallothinin, phosphatase, Snai1, TRAF 6. The function control agent according to claim 6, wherein the function control agent is at least one selected from the group consisting of P axi 1 1 in, Z yxin and J ade-1.

8. The function control agent according to claim 1, wherein the substance capable of controlling the zinc ion concentration is a substance that regulates expression and Z or function of a zinc ion transporter.

9. The function control agent according to claim 8, wherein the zinc ion transporter is at least one selected from the group consisting of human Z IPs and human CDFs containing an LI V family.

10. Antigen-presenting cells are dendritic cells, macrophages, Langerhans cells, B cells, The function control agent according to any one of claims 1 to 9, which is at least one selected from the group consisting of thymic epithelial cells, synoviocytes, vascular endothelial cells, and keratinocytes.

1 1. The function control agent according to claim 10, wherein the antigen-presenting cell is a rod-shaped cell.

1 2. A prophylactic / therapeutic agent for a disease involving the immune system, comprising the function control agent according to any one of claims 1-11.

1 3. The disease involving the immune system is selected from the group consisting of self-immune disease, allergic disease, rejection at the time of organ transplantation, decreased immune response due to infection and tumor formation, and abnormal immune response at the time of vaccine use The preventive / therapeutic agent according to claim 12, which is at least one kind. -

14. A prophylactic / therapeutic drug for diseases involving an immune system, containing as an active ingredient a substance that can control the intracellular zinc ion concentration.

1 5. The prophylactic / therapeutic agent according to claim 14, wherein the substance capable of controlling the zinc ion concentration is zinc ion.

1 6. The preventive / therapeutic agent according to claim 15, wherein the zinc ion is introduced into the cell by a zinc ionophore.

1 7. The prophylactic / therapeutic agent according to claim 14, wherein the substance capable of controlling the zinc ion concentration is a zinc ion chelator.

1 8. Zinc ion chelator is 2,3-dimercapto-1-propanesulfonic acid (DMP S), N, N, Ν ', N' —tetrakis (2-pyridylmethyl) ethylene diamine (T PEN), ethylene diamine (EDTA) The prophylactic / therapeutic agent according to claim 17, which is at least one selected from the group consisting of N) (6-methoxyl 8-quinolyl) p-toluenesulfonamide (TSQ).

1 9. The prophylactic / therapeutic agent according to claim 14, wherein the substance capable of controlling the zinc ion concentration is a substance that regulates the expression and / or function of a zinc ion-requiring protein.

20. Zinc ion-requiring protein is R af — 1, PKC a, GEF—H l, H DAC, SQSTM1, ubiquitin-protein ligase E 3, ubiquitin-coupled protein E 2, metamouthoneone, phosphatase, S nai 1 , TRAF 10. The prophylactic / therapeutic agent according to claim 19, which is at least one selected from the group consisting of 6, P axi 1 1 in, Z yxin and J ade-1 force.

2 1. The prophylactic / therapeutic agent according to claim 14, wherein the substance capable of controlling the zinc ion concentration is a substance that regulates expression and Z or function of a zinc ion transporter.

22. The prophylaxis / treatment according to claim 21, wherein the zinc ion transporter is at least one selected from the group consisting of human Z IPs and human CDFs containing LI family.

23. Diseases involving the immune system are selected from the group consisting of autoimmune diseases, allergic diseases, rejection at the time of organ transplantation, reduced immune response due to infection and tumor formation, and abnormal immune response when using acupuncture The prophylactic / therapeutic agent according to any one of claims 14 to 22, which is at least one kind.

24. The antigen-presenting cell is at least one selected from the group consisting of dendritic cells, macrophages, Langerhans cells, B cells, thymic epithelial cells, synovial cells, vascular endothelial cells, and keratinocytes. The preventive / therapeutic drug according to any one of 1.

25. The preventive / therapeutic agent according to claim 24, wherein the antigen-presenting cell is a dendritic cell.

26. A method of controlling the function of an antigen-presenting cell, comprising controlling the concentration of zinc ions in a cell in vitro.

27. The method according to claim 26, wherein the zinc ion concentration is controlled by introducing zinc ions into cells.

28. The method according to claim 27, wherein the zinc ion is introduced into the cell by a zinc ionophore.

29. The method according to claim 26, wherein the zinc ion concentration is controlled by a zinc ion chelator.

30. Zinc ion chelator is 2,3-dimercapto _ 1 _propanesulfonic acid (DMP S), Ν, Ν, Ν ', Ν'-Tetrakis (2-pyridylmethyl) ethylene diamine (ΤΡ ΕΝ), ethylene diamine (EDTA) ) And Ν— (6-Methoxy The method according to claim 29, which is at least one member selected from the group consisting of 8-quinolyl) 1-toluenesulfonamide (TSQ).

3. The method according to claim 26, wherein the zinc ion concentration is controlled by regulating the expression or function of a zinc ion-requiring protein.

32. Zinc ion-requiring protein is R af — l, PKC a, GEF— H l, H DAC, SQSTM1, ubiquitin monoprotein ligase E 3, ubiquitin conjugate protein E 2, metallothionein, phosphatase, S nai 1, TRAF 6, The method according to claim 31, wherein the method is at least one selected from the group consisting of P axi 1 1 in, Z yxin and J ade-1.

33. The method of claim 26, wherein the zinc ion concentration is controlled by adjusting the expression and Z or function of the zinc ion transporter.

34. The method according to claim 33, wherein the zinc ion transporter is at least one selected from the group consisting of human Z IPs and human CD Fs containing L I V family.

35. The antigen-presenting cell is at least one selected from the group consisting of rod-shaped cells, macrophages, Langerhans cells, B cells, thymic epithelial cells, synovial cells, vascular endothelial cells, and keratinocytes. The method according to any one of the above.

36. A screening method for a compound capable of controlling the function of antigen-presenting cells, comprising measuring intracellular zinc ion concentration.

37. The method according to claim 36, wherein the antigen-presenting cell is at least one selected from the group consisting of dendritic cells, macrophages, Langerhans cells, B cells, thymic epithelial cells, synovial cells, vascular endothelial cells, and keratinocytes. .

38. The method according to claim 37, wherein the antigen-presenting cell is a dendritic cell.

39. Use of a substance capable of controlling the zinc ion concentration in the cell wall to produce a function control agent for antigen presenting cells.

40. Use of a function-controlling agent according to any one of claims 1 to 11 for the manufacture of a prophylactic agent for a disease associated with an immune system.

4 1. Prevention of diseases involving the immune system ■ Use of substances that can control the concentration of intracellular zinc ions to produce therapeutic drugs.